检测原理
试剂盒采用双抗体夹心法酶联免疫吸附试验(ELISA)。往预先包被植物玉米矮花叶病毒(MDMV)捕获抗体的包被微孔中,依次加入阴性对照、阳性对照、样本、HRP标记的检测抗体,经过温育并*洗涤。用底物TMB显色,TMB在过氧化物酶的催化下转化成蓝色,并在酸的作用下转化成zui终的黄色。颜色的深浅和样品中的植物玉米矮花叶病毒(MDMV)呈正相关。用酶标仪在450nm 波长下测定吸光度(OD 值),判断样品是否含有植物玉米矮花叶病毒(MDMV)。
样品收集、处理及保存方法
1. 血清:使用不含热原和内毒素的试管,操作过程中避免任何细胞刺激,收集血液后,3000转离心10分钟将血清和红细胞迅速小心地分离。
2. 血浆:EDTA、柠檬酸盐或肝素抗凝。3000转离心30分钟取上清。
3. 细胞上清液:3000转离心10分钟去除颗粒和聚合物。
4. 组织匀浆:将组织加入适量生理盐水捣碎。3000转离心10分钟取上清。
5. 保存:如果样本收集后不及时检测,请按一次用量分装,冻存于-20℃,避免反复冻融,在室温下解冻并确保样品均匀地充分解冻。
自备物品
操作注意事项
试剂盒组成
名称 |
96孔配置 |
48孔配置 |
备注 |
微孔酶标板 |
96孔 |
48孔 |
无 |
阴性对照 |
0.3mL |
0.3mL |
无 |
阳性对照 |
0.3mL |
0.3mL |
无 |
样本稀释液 |
6mL |
3mL |
无 |
检测抗体-HRP |
10mL |
5mL |
无 |
20×洗涤缓冲液 |
25mL |
15mL |
按说明书进行稀释 |
底物A |
6mL |
3mL |
无 |
底物B |
6mL |
3mL |
无 |
终止液 |
6mL |
3mL |
无 |
封板膜 |
2张 |
2张 |
无 |
说明书 |
1份 |
1份 |
无 |
自封袋 |
1个 |
1个 |
无 |
试剂的准备
20×洗涤缓冲液的稀释:蒸馏水按1:20稀释,即1份的20×洗涤缓冲液加19份的蒸馏水。
洗板方法
操作步骤
结果判断
1. 试验有效性:阳性对照孔OD值平均值≥1.00;
阴性对照孔OD值平均值≤0.15。
2. 临界值(Cut off)计算:临界值=阴性对照孔平均值+0.15
3. 阴性判断:样品OD值<临界值(Cut off),样品为阴性
4. 阳性判断:样品OD值>临界值(Cut off),样品为阳性
试剂盒性能
免责声明
FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
PlantMaize Dwarf Mosaic Virus(MDMV)ELISA Kit instruction
Intended use
ThisMDMVELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures.In order todetermine whether containsMDMVin the sample, thisMDMVELISA Kit includes Negative controlandPositive control. The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. The color depth was positively correlated with theMDMV in the sample .
Samplecollection and storages
Serum- Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for10 minutes at approximately3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles
Plasma- Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for30minutes at3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Cell culture supernates and other biological fluids-Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Note:The samples shouldbe centrifugatedadequately and no hemolysis or granule was allowed.
Materials required but not supplied
1.Standard microplate reader(450nm)
2.Precision pipettes and Disposable pipette tips.
3.37 ℃ incubator
Precautions
1.Donotsubstitutereagentsfromone kitto another.Standard, conjugateandmicroplates arematchedfor optimal performance.Useonlythereagentssuppliedbymanufacturer.
2.Donotremovemicroplatefromthestorage baguntilneeded.Unusedstripsshouldbestored at2-8°Cintheir pouchwiththe desiccantprovided.
3.Mix all reagents before using.
Remove allkitreagentsfromrefrigerator and allowthemtoreachroomtemperature( 20-25°C)
Materials supplied
Name |
96determinations |
48determinations |
Microelisa stripplate |
96 strips |
48 strips |
Negative control |
0.3ml |
0.3ml |
Positive control |
0.3ml |
0.3ml |
Sample diluent |
6.0ml |
3.0ml |
HRP-Conjugate reagent |
10.0ml |
5.0ml |
20X Wash solution |
25ml |
15ml |
Chromogen Solution A |
6.0ml |
3.0ml |
Chromogen Solution B |
6.0ml |
3.0ml |
Stop Solution |
6.0ml |
3.0ml |
Closure plate membrane |
2 |
2 |
User manual |
1 |
1 |
Sealed bags |
1 |
1 |
Reagent preparation
20×wash solution:Dilute withDistilled or deionized water1:20.
Assay procedure
1.Prepare allreagentsbeforestartingassayprocedure.ItisrecommendedthatallStandardsand Samplesbe addedin duplicatetotheMicroelisaStripplate.
2.Separately add Positive control and Negative control 50μl to the Positive and Negative well,Add testing sample10μlThen addsample diluent40μl to testing sample well; Blank welldoesn’t add anyting.
3.Add100μlofHRP-conjugate reagentto each well,cover with anadhesive stripandincubatefor60 minutesat37°C.
4.Aspirate each well and wash, repeating the processfourtimes for a total of fivewashes.Wash by filling each well with WashSolution(400μl) using a squirt bottle, manifolddispenseror autowasher. Complete removal of liquid at each step is essential to goodperformance. After the last wash, remove any remaining WashSolutionby aspirating ordecanting. Invert the plate and blot it against clean paper towels.
5.Add chromogen solution A 50μl and chromogen solution B 50μl to each well.Gently mixandincubate for 15 minutes at37°C.Protect from light.
6.Add 50μlStop Solution to each well. The color in the wells should change from blue toyellow. If the color in the wells is green or the color change does not
appear uniform,gently tap the plate to ensure thorough mixing.
7.ReadtheOpticalDensity(O.D.)at450nmusingamicrotiterplatereaderwithin15minutes.
Determine the result
1.Test validity: the average of Positive control well≥1.00; the average of Negative control well ≤0.15.
2.Calculate Critical(CUT OFF): Critical= the average of Negative controlwell + 0.15.
NegativeResult: sample OD< Calculate Critical(CUT OFF) is Negative.
PositiveResult:sample OD≥ Calculate Critical(CUT OFF) is Positive.
Storage and validity
Storage:2-8℃.
validity: six months.
FOR RESEARCH USE ONLY;
NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!
PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!